Multi‐tool monitoring: integrating non‐invasive methods to assess vertebrate richness, functional diversity and trophic complexity

Effective biodiversity monitoring is essential for understanding taxonomic, functional and trophic dynamics in changing ecosystems. Non-invasive tools such as environmental DNA (eDNA) metabarcoding, passive acoustic monitoring (PAM) and camera trapping offer complementary strengths, yet their relative effectiveness for assessing biodiversity, in particular functional diversity and trophic structure, remains underexplored. These approaches are particularly valuable in rewilding landscapes, where restoration actions create heterogeneous, rapidly changing habitats that require scalable monitoring solutions. We applied an integrated monitoring approach at a rewilding site in Scotland, comparing vertebrate diversity detected by metabarcoding four eDNA sample types (water, soil, tree rolling and scat) with PAM and camera traps. Each method was evaluated for taxonomic richness, community composition, functional diversity via trait-based analyses and trophic complexity through co-occurrence networks, metrics rarely assessed together across methods. In total, 79 vertebrate taxa were detected: PAM captured 52 taxa, eDNA 44 and camera traps 12. Of the eDNA substrates, water and tree-rolling samples had the highest richness. Each method detected unique species, with birds and bats best covered by PAM, and small mammals by eDNA. Community composition varied between methods, with eDNA substrates capturing broader communities. PAM revealed the strongest community differences between wooded and open habitats. Tree rolling and water eDNA captured the greatest functional diversity, while PAM showed higher redundancy. No single method captured all functional traits. Integrating eDNA methods with PAM produced the most complete trophic network. PAM provided the most complete network but missed key terrestrial interactions that were filled by eDNA. Practical implication. Rewilding and restoration programmes require an integrated multi-method 'toolkit' to comprehensively capture vertebrate richness, functional diversity and trophic complexity. We recommend combining PAM with either tree rolling or water eDNA sampling as an optimal monitoring strategy, balancing their complementary strengths in taxonomic and functional coverage.